Cigarette smoke-induced reduction in binding of the salivary translocator protein is not mediated by free radicals.

R. Nagler, D. Savulescu, M. Gavish

Department of Neuroscience, Faculty of Medicine, Rappaport Family Institute for Research in the Medical Sciences, Technion – Israel Institute of Technology, 31096, Haifa, Israel



Oral cancer is the most common malignancy of the head and neck and its main inducer is exposure to cigarette smoke (CS) in the presence of saliva. It is commonly accepted that CS contributes to the pathogenesis of oral cancer via reactive free radicals and volatile aldehydes. The 18 kDa translocator protein (TSPO) is an intracellular receptor involved in proliferation and apoptosis, and has been linked to various types of cancer. The presence of TSPO in human saliva has been linked to oral cancer, and its binding affinity to its ligand is reduced following exposure to CS. In the present study we wished to further investigate the mechanism behind the CS-induced reduction of TSPO binding by exploring the possible mediatory role of reactive oxygen species (ROS) and volatile aldehydes in this process. We first analyzed TSPO binding in control saliva and in saliva exposed to CS in the presence and absence of various antioxidants. These experiments found that TSPO binding ability was not reversed by any of the antioxidants added, suggesting that CS exerts its effect on TSPO via mechanisms that do not involve volatile aldehydes and free radicals tested. Next, we analyzed TSPO binding in saliva following addition of exogenous ROS in the form of H2O2. These experiments found that TSPO binding was enhanced due to the treatment, once again showing that the CS-induced TSPO binding reduction is not mediated by this common form of ROS. However, the previously reported CS-induced reduction in salivary TSPO binding together with the role of TSPO in cells and its link to cancer strongly suggest that TSPO has a critical role in the pathogenesis of CS-induced oral cancer. The importance of further elucidating the mechanisms behind it should be emphasized.

Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM).

KEYWORDS: 18 kDa translocator protein (TSPO); Aldehydes; Cigarette smoke; Free radicals; Oral cancer; Saliva

PMID: 26582415



Cigarette smoke (CS) plays a major role in the pathogenesis of oral cancer, containing at least 400 proven carcinogens [1]. Constant exposure of the oropharyngeal mucosa to these carcinogens is believed to result in oxidative damage, leading to cellular mutagenesis, which may then contribute to cancer development. CS carcinogens include un-complex metals, free radicals such as reactive oxygen species (ROS) and volatile aldehydes [2]. Highly reactive hydroxyl free radicals, along with aldehydes have previously been suggested to be major mediators of the mutagenic effect of CS in the oral cavity [3]. Translocator protein (TSPO) is an intracellular protein involved in major cellular processes such as proliferation and apoptosis [4, 5]. We have previously reported the presence of TSPO in human saliva, and found that its expression is enhanced in oral cancer tissues [6]. Additionally, we found that following exposure to CS, TSPO binding affinity to its ligand is reduced both in vivo and in vitro [6]. TSPO has previously been suggested to play a protective role against ROS cellular damage [7, 8]. The TSPO protein levels have been shown to correlate to resistance to toxicity caused by the reactive oxygen species H2O2 in hematopoietic cells, [9], and TSPO polymers have been shown to be produced in response to ultra-violet irradiation-induced ROS in cancer cells [10]. These findings, together with ours, have led us to explore the possible role of free radicals in the CS-induced reduction in TSPO binding affinity.

We hypothesized that free radicals at least partially mediate the CS-induced reduction in TSPO binding affinity. In order to test our hypothesis, we designed two sets of experiments. In the first set we tested TSPO binding following addition of various antioxidants to saliva prior to CS exposure. Antioxidants act as free radical scavengers (11) and we were interested to investigate whether they would be able to prevent, at least in a partial manner, the TSPO binding reduction caused by CS. Each antioxidant used in our assay targets a different form of free radicals previously reported to be present in CS, as follows: the aldehyde scavenger, glutathione (GSH) [3, 12], the cyanide chelator, hydroxocobalamine [13, 14], the iron chelator, deferoxamine (desferal) [3], and the copper chelator, penicillamine [15]. We postulated that if we add antioxidants to saliva samples prior to CS exposure, they would react with CS-borne reactive free radicals, therefore preventing any effect that these radicals might have on TSPO binding. Our second set of experiments was based on a more direct approach, which consisted of addition of exogenous H2O2 to saliva for 60 minutes prior to analysis of TSPO binding and cell viability. We postulated that this experiment would mimic the effect of CS on TSPO binding.

Our first set of experiments resulted in a ~30% reduction in TSPO binding in all CS-exposed samples as compared to their controls, with no regards to the presence of any antioxidant tested. Moreover, in saliva samples exposed to either hydroxocobalamine or glutathione there was an overall increase in TSPO binding both in control and CS-treated samples, as compared to non-treated samples. In the second set of experiments, exposure of saliva samples to H2O2 led to a statistically significant 66% increase in TSPO binding, as compared to control (p ¼ 0.0002). Together, our findings indicate that in spite of the extensive oxidative stress caused to the salivary cells following exposure to CS, as previously reported [6], copper, iron and cyanide radicals, as well as H2O2 and aldehydes do not appear to be involved in the inhibitory effect of CS on TSPO binding. Moreover, aldehydes and cyanide radicals seem to induce an increase in TSPO binding, which is contradictory to the reduction in the protein’s binding caused by CS.

In summary, the first set of findings in this study demonstrate that addition of specific scavengers for copper, iron, aldehydes and cyanide to saliva prior to CS exposure does not alter the effect of CS on TSPO binding. This strongly suggests that aldehydes, as well as copper, iron and cyanide radicals present in CS are not involved in the molecular mechanism behind the CS-induced reduction in TSPO binding. It is therefore possible that other free radicals present in CS mediate the effect of CS on TSPO binding.  This is further supported by data we have recently published, where addition of free radical-containing saliva to the medium of lung cancer cells did not affect TSPO binding in the presence or absence of CS [16]. We also demonstrate here that addition of glutathione or hydroxocobalamine to saliva prior to CS exposure induces an increase in basic levels of TSPO binding with no regards to CS exposure. We suggest that there is a basic amount of reactive aldehydes and cyanide radicals present in saliva, which might directly or indirectly inhibit TSPO binding to a certain extent, resulting in the levels observed in control samples. Once chelators for these oxidants are added, they are able to block them, thereby removing their inhibitory effect on TSPO binding. However, since these chelators do not change the effect of CS exposure on TSPO binding, it seems that any additional, CS-derived aldehydes and cyanide radicals do not affect TSPO binding. Our second set of experiments adds a further layer of complexity, with H2O2 leading to an effect on TSPO binding opposite than that of CS.  Our data presented here is not sufficient for fully understanding the effects of the different free radicals on TSPO binding, but we do propose a scenario which might be able to partially explain our findings: some CS-borne free radicals might induce an increase in TSPO binding, while others have the opposite effect. This would suggest that during CS exposure salivary components are subject to attack by different CS borne agents that can lead to opposite effects. The balance between such agents would then define the final outcome, which in the case of TSPO is reduction in binding ability. As previously reported [6], reduced TSPO binding is also seen in vivo, in saliva of heavy smokers when compared to non-smokers. This might be a result of modifications in the TSPO-encoding gene, as well as conformational changes in the protein itself. In the current study, we exposed the saliva samples to antioxidants, H2O2 and CS for short periods of time, and therefore we expect all modifications in TSPO binding to be caused mainly on the protein level. We postulate that CS-borne agents different than the ones tested here, interact with TSPO, thus causing a change in its conformation, which compromises its ability to bind ligands. Despite the fact that the free radicals tested here were either not found to be involved in TSPO binding reduction, or even causing an increase in it, we do expect that they would be involved in previously reported long term DNA modifications [12,17].



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